Process for 3β-aminoazetidin-2-ones

ABSTRACT

1-[α-(Carboxy)-4-hydroxybenzyl]-3β-aminoazetidin-2-one esters are prepared by converting 2-acyl-3,3-dialkyl-7-oxo-α-[4-(benzyloxy)phenyl]-4-thia-2,6-diazabicyclo[3.2.0]-heptane-6-acetic acid esters with mercuric acetate in an aqueous organic solvent mixture, e.g., in aqueous methanol, to 7-oxo-3-phenyl-α-[4-(benzyloxy)phenyl]-4-oxa-2,6-diazabicyclo[3.2.0]hept-2-ene-1-acetic acid esters and the latter are reacted with PCl 5  and pyridine to provide the monocyclic 1-[α-(carboxy)-4-benzyloxybenzyl]-3β-(α-chlorobenzylideneamino)-4-chloroazetidin-2-one esters. Reduction of the dichloro azetidin-2-one with an organo tin hydride and azobisisobutyronitrile affords the deschloro, 1-[α-(carboxy)-4-benzyloxybenzyl]-3β-benzylideneaminoazetidin-2-one ester. The latter is hydrolyzed and the 4-benzyloxy group is cleaved via catalytic hydrogenolysis to yield an ester of 1-[α-(carboxy)-4-hydroxybenzyl]-3β-aminoazetidin-2-one. The 3β-amino ester is useful for the preparation of the antibiotic FR 1923 (nocardicin).

BACKGROUND OF THE INVENTION

This invention is concerned with a chemical process for preparingsubstituted azetidin-2-ones. In particular it is concerned with aprocess for the preparation of certain 3β-aminoazetidin-2-ones which areuseful intermediates in the synthesis of azetidin-2-one antibiotics forexample the antibiotic FR 1923.

The antibiotic FR 1923, also referred to as nocardicin, has beenpreviously described, for example in Belgium Pat. No. 830,934 and by H.Aoki et al., 15th Interscience Conference on Antimicrobial Agents andChemotherapy, Abstract No. 97, Sept. 1975. Nocardicin has the followingstructural formula. ##STR1##

Antibiotic FR 1923 has been obtained by culturing Nocardia uniformisvar. Tsuyamanensis ATCC 21806 as described by U.S. Pat. No. 3,923,977issued Dec. 2, 1975.

This invention provides a process for preparing the "nucleus" ofnocardicin represented by the following formula, ##STR2## and esters andhydroxy group derivatives thereof.

The nocardicin nucleus is useful in the preparation of nocardicin viaacylation with an amino-protected and esterified derivative of4-(3-amino-3-carboxypropoxy)phenylglyoxylic acid, the acyl side chain ofnocardicin. The acylation product, a 3β-phenylglyoxamide azetidin-2-oneis deblocked and de-esterified and is converted to nocardicin byformation of the oxime derivative. The synthesis of the acyl side chainof nocardicin and the amino-protected esters thereof is described inco-pending application Ser. No. 739,160 now abandoned filed Nov. 5, 1976and its continuing application Ser. No. 825,344 filed Aug. 17, 1977.

DETAILED DESCRIPTION

According to the process of this invention, the fused bicyclic"thiazolidine-azetidinone" represented by the formula 1 ##STR3## whereinR is C₁ -C₃ alkyl, phenyl, or benzyl; both of R' are methyl or ethyl; R₁is methyl, benzyl, 4-methoxybenzyl, or diphenylmethyl; and R₂ is benzyl,4-methoxybenzyl, or diphenylmethyl; is reacted with mercuric acetate inan aqueous organic solvent to provide the fused bicyclic"oxazoline-azetidinone" represented by the formula 2. ##STR4## whereinR, R₁, and R₂ have the same meanings as defined above.

The oxazoline-azetidinone is reacted with phosphorus pentachloride inthe presence of pyridine to effect the opening of the oxazoline ring andprovide the chlorinated 3β-iminoazetidin-2-one represented by theformula 3. ##STR5##

Reduction of (3) via a free radical reduction with an organotin hydride,eg. a trialkyltin hydride, and azobisisobutyronitrile affords the deschloro-imino-azetidin-2-one represented by the formula 4. ##STR6##

The 3β-iminoazetidin-2-one (4) is hydrolyzed to the3β-aminoazetidin-2-one represented by the formula 5. ##STR7##

The thiazolidine azetidinone represented by the formula 1 which is thestarting material employed in the process of this invention is namedformally as a 2-acyl-3,3-dimethyl (or diethyl)-7-oxo-α-[4-(benzyloxy,p-methoxybenzyloxy ordiphenylmethyloxy)phenyl]-4-thia-2,6-diazabicyclo[3.2.0]heptane-6-aceticacid, methyl, benzyl, 4-methoxybenzyl, or diphenylmethyl ester. Forconvenience of description, the compounds represented by the formula 1are referred to herein as thiazolidine-azetidinones. The preparation ofthese thiazolidine-azetidinones will be described hereinafter.

In carrying out the process of this invention, thethiazolidine-azetidinone is reacted with mercuric acetate in an aqueouswater-miscible organic solvent at a temperature between about 25° C. andabout 100° C. to form the oxazoline-azetidinone represented by theformula 2. Between about 1 mole and about 4 moles of mercuric acetateper mole of thiazolidine azetidinone is a suitable ratio of reactants,although excess amounts of mercuric acetate can be used. Preferably, 2moles of mercuric acetate per mole of starting material are used.

Water-miscible organic solvents which can be used include the etherssuch as tetrahydrofuran, dioxane, and the dimethyl ether of ethyleneglycol; the lower alcohols such as methanol and ethanol; and thenitriles such as acetonitrile. The amount of water in solution with theorganic solvent is not critical and in general between about 10 percentand about 60 percent water by volume is a suitable amount. The preferredaqueous solvent is aqueous tetrahydrofuran containing between about 10and 60 percent water.

The reaction can be carried out at room temperature or with heating atsteam bath temperatures.

In performing the process of this invention, it is preferable to obtainthe oxazoline-azetidinone relatively free of contaminants prior to itsuse in the succeeding step of the process. Accordingly, following thereaction, the reaction mixture is filtered to remove insolubles and thefiltrate is extracted with a water immiscible organic solvent. Solventssuch as ethyl acetate, amyl acetate, chloroform, or methylene chlorideare suitable extractants. The extract is washed, dried, and evaporatedto provide the oxazoline-azetidinone as a residue suitable for use inthe next reaction of the process. Alternatively, the product can beobtained in crystalline form by crystallization from a suitable solventsuch as diethyl ether.

The oxazoline-azetidinone formed as product in the above-describedreaction is represented by the foregoing formula 2 and is formally nameda 7-oxo-3-alkyl(phenyl or benzyl)-α-[4-benzyloxy, p-methoxybenzyloxy ordiphenylmethyloxy)phenyl]-4-oxa-2,6-diazabicyclo[3.2.0]hept-2-ene-1-aceticacid, methyl, benzyl, 4-methoxybenzyl, or diphenylmethyl ester.

The oxazoline-azetidinone obtained as described above is chlorinatedwith a phosphorus or antimony chloride to provide the dichloro compound,a 1-[α-(benzyloxycarbonyl, methoxycarbonyl, 4-methoxycarbonyl, ordiphenylmethoxycarbonyl)-4-benzyloxy, 4-methoxybenzyloxy, ordiphenylmethoxybenzyl]-3β-(α-chloroimino)-4α-chloroazetidin-2-onerepresented by the foregoing formula 3.

The chlorination is carried out under anhydrous conditions withphosphorus pentachloride or phosphorus trichloride in an inert solvent.Antimony tri or pentachloride can also be used as the chlorinatingagent, however, phosphorus pentachloride is preferred. The chlorinationcan be carried out at a temperature between about -10° C. and about 45°C. and preferably at room temperature of about 20°-25° C.

"Inert solvents" are solvents which are unreactive under thechlorination conditions of the process, for example, the chlorinatedhydrocarbon solvents such as chloroform, methylene chloride,dichloroethane, and trichloroethane are suitable inert solvents.

The reaction is carried out in the presence of a tertiary organic aminewhich serves as a hydrogen chloride acceptor. Tertiary amines such aspyridine, the methylated pyridines, quinoline, or the tertiaryalkylamines, for example, triethylamine can be used. Pyridine ispreferred.

The phosphorus or antimony chloride is employed in excess, for example,between about a tenth molar and 2 molar excess per mole ofoxazoline-azetidinone. The tertiary amine, for example, the preferredpyridine is employed in an amount equimolar with the phosphorus orantimony chloride.

The reaction is carried out by adding the chloride to a solution of theoxazoline-azetidinone in the inert solvent. The tertiary amine is addedand the reaction mixture is agitated by stirring or shaking until thereaction is completed. The dichloro product is recovered by diluting thereaction mixture with water or brine below room temperatures andpreferably about 0°-5° C. and separating the organic layer containingthe product. The organic layer is washed, dried, and evaporated toprovide the dichloro product as a residue. The product can be furtherpurified; however, further purification is unnecessary in the process ofthis invention.

The 3β-(α-chloroimino)-4α-chloroazetidin-2-one, product of thechlorination, is subjected to reduction with an organo tin hydride underfree radical reduction conditions initiated with azobisisobutyronitrile.The reduction effects the replacement of both chlorine atoms withhydrogen atoms to provide the des chloro azetidin-2-one reductionproduct, a 1-[α-(methyl, benzyl, 4-methoxybenzyl, ordiphenylmethoxycarbonyl)-4-benzyloxy, 4-methoxybenzyloxy, ordiphenylmethoxybenzyl]-3β-benzylidene (or alkylidene)aminoazetidin-2-onerepresented by the formula 4.

The reduction is carried out in an inert solvent under essentiallyanhydrous conditions. Aromatic hydrocarbon solvents such as benzene,toluene, and the xylenes provide a suitable medium for the reaction.Toluene is a preferred solvent.

Organo tin hydrides which can be employed in the process are representedby the following formula ##STR8## wherein R₃, R₄, and R₅ independentlyare C₁ -C₄ alkyl, phenyl, or phenyl substituted by methyl or chloro.

Examples of such tin hydrides are the trialkyl tin hydrides such astri(n-butyl)tin hydride, tin(n-propyl)tin hydride, trimethyltin hydride,and triethyltin hydride; the triaryltin hydrides such as triphenyltinhydride, and tri(p-tolyl)tin hydride; and the mixed alkyl and mixedalkyl aryltin hydrides such as di(n-butyl)phenyltin hydride,dimethylethyltin hydride, and diphenylmethyltin hydride.

A preferred tin hydride is tri(n-butyl)tin hydride.

The organo tin hydride is employed in a molar ratio of 2:1, i.e., 2moles of organo tin hydride per mole of3β-(α-chloroimino)-4α-chloroazetidin-2-one. A slight excess of the tinhydride can be used without deleterious effect on the reaction productand may be used where trace amounts of water may be present.

The azobisisobutyronitrile, formally named2,2'-azobis(2-methylpropionitrile) and represented by the followingstructural formula ##STR9## is employed in the reduction in an amountequimolar with the tin hydride.

The reaction is carried out at a temperature between about 65° C. andabout 85° C. and preferably at about 70° C.

The reaction is performed by adding the organo tin hydride and theazobisisobutyronitrile to a solution of the3β-(α-chlorobenzylideneamino) or3β-(α-chloroalkylideneamino)-4α-chloroazetidin-2-one in the dry aromatichydrocarbon, for example, toluene at or about room temperature. Freshlydistilled solvents are preferred. After addition is complete, thereaction mixture is heated to a temperature between about 65° C. andabout 85° C. with stirring. During the reaction, it is preferable toexclude atmospheric moisture by maintaining the mixture in an atmosphereof nitrogen. The course of the reaction can be followed by thin layerchromatography. When the reaction is complete, the mixture is dilutedwith an organic water-immiscible solvent such as ethyl acetate and iswashed with dilute base and water, is dried, and evaporated to providethe 3β-benzylideneaminoazetidin-2-one or alkylideneaminoazetidin-2-one(formula 4). The product can be obtained crystalline from a suitablesolvent, for example, toluene or benzene, on cooling.

In the last step of the process of this invention, the3β-iminoazetidin-2-one is converted under acidic conditions to the3β-aminoazetidin-2-one represented by the formula 5. The3β-benzylideneaminoazetidin-2-one or 3β-alkylideneaminoacetidin-2-oneemployed in the acid removal of the benzal or alkylidene group can bepurified crystalline material obtained in the organo tin hydridereduction step or crude material. In the final step of the process, itis preferable to employ the crude 3β-benzylideneamino (oralkylideneamino)azetidin-2-one obtained in the tin hydride reduction asdescribed above. The 3β-aminoazetidinone (5) is obtained as the saltformed with the acid employed.

Acids which can be used include the mineral acids such as hydrochloricacid, sulfuric acid, or phosphoric acid and the organic sulfonic acidssuch as the lower alkylsulfonic acids, for example, methanesulfonicacid, ethanesulfonic acid, and propanesulfonic acid; the aromaticsulfonic acids such as benzenesulfonic acid, the toluenesulfonic acids,and α or β-naphthylenesulfonic acid. Preferred acids are hydrochloricacid and p-toluenesulfonic acid.

In carrying out the reaction, the 3β-iminoazetidin-2-one (4) isdissolved in a water immiscible organic solvent such as an ester, forexample, ethyl acetate or amyl acetate; a chlorinated hydrocarbon, forexample, methylene chloride or trichloroethane; and the solution isshaken with excess hydrochloric acid, e.g., 1N-hydrochloric acid. Theorganic phase is separated, dried, and evaporated to dryness to providethe 3β-aminoazetidin-2-one (5) hydrochloride. The product can bepurified by trituration with petroleum ether or by recrystallization.

Alternatively, the benzal or alkylidene group of (4) can be removed toprovide the 3β-aminoazetidin-2-one in the following manner. The crudetin hydride reduction product (4) is dissolved in diethyl ether and thesolution is cooled to about 0°-5° C. in an ice-water mixture. Hydrogenchloride is bubbled through the cold solution with stirring. Thesolution is then allowed to warm to room temperature with continuedstirring. The solution is evaporated to dryness in vacuo and the residueof 3β-aminoazetidinone hydrochloride is purified by trituration withdiethyl ether or petroleum ether.

Preferably, in the process of this invention, crude (4) is converted to(5) with p-toluenesulfonic acid. For example, (4) is dissolved in ethylacetate or other suitable solvent and a slight excess ofp-toluenesulfonic acid monohydrate is added to the solution. The benzalgroup is rapidly removed as shown by the disappearance of the imine viathin layer chromatography (silica gel, benzene:ethyl acetate, 7:3). Onstanding, or with cooling, the p-toluenesulfonic acid salt of the3β-aminoazetidin-2-one forms as a crystalline precipitate.

The 3β-aminoazetidin-2-one salts are readily converted to the 3β-aminocompound (5) as the free amine as follows. The salt is dissolved in asuitable water immiscible solvent, for example, ethyl acetate and thesolution is shaken vigorously with an aqueous solution of a base such assodium or potassium bicarbonate or sodium or potassium carbonate. Theorganic layer is separated, dried, and evaporated to provide the freeamine (5) as a residue. The 3β-aminoazetidin-2-one obtained is generallyof sufficient quality for use in the preparation of nocardicin asdescribed hereinafter. Thus, in the process of this invention, the crudeorgano tin hydride reduction product (4) is converted to the 3β-aminocompound (5) via the acid salt. The direct preparation of the salt formprovides for the purification of the 3β-aminoazetidin-2-one nucleus (5).Should further purification of (5) be necessary, it can be achieved bychromatography over silica gel.

Representative of the 3β-aminoazetidin-2-ones provided by the process ofthis invention are1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one,1-[α-(methoxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one,1-[α-(4-methoxybenzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one,1-[α-(diphenylmethoxycarbonyl)-4-diphenylmethoxybenzyl]-3β-aminoazetidin-2-one,1-[α-(4-methoxybenzyloxycarbonyl)-4-diphenylmethyloxybenzyl]-3β-aminoazetidin-2-oneand1-[α-(diphenylmethoxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one.

As mentioned previously, the starting material employed in the processis the bicyclic thiazolidine-azetidinone represented by the foregoingformula 1. The thiazolidine-azetidinone is prepared as described incopending application Ser. No. 739,161 filed Nov. 5, 1976.

According to the described process a2,2-dialkyl-3-acylthiazolidine-4-carboxylic acid having theL-configuration is reacted with an ester of a 4-hydroxy-protectedD-phenylglycine to obtain the corresponding amide (A) as shown in thefollowing reaction scheme. ##STR10##

In the above formulas R, R₁, R₂, and R' have the same meanings aspreviously defined.

The preparation of the amide (A) is carried out by reacting the activeester of the thiazolidine-4-carboxylic acid formed with1-hydroxybenzotriazole with the esterified and hydroxy-protectedphenylglycine in the presence of dicyclohexylcarbodiimide.

The thiazolidine amide (A) is converted to the cyclicthiazolidine-azetidinone represented by the formula 1 as shown in thefollowing scheme. ##STR11##

As shown in the above reaction scheme, the thiazolidine amide is firstconverted to the 5α-benzoate derivative (B) by reacting (A) with benzoylperoxide. The reaction is carried out by heating the amide in an inertsolvent with benzoyl peroxide. Suitable solvents include the hydrocarbonsolvents such as benzene and toluene, or the chlorinated hydrocarbonsolvents such as methylene chloride and chloroform. An excess of benzoylperoxide is employed and preferably between about a 2 and 4 molarexcess.

The 5α-benzoate (B), which can be purified and separated from unreactedstarting material by chromatography over silica gel, is then reactedwith hydrogen chloride in an inert solvent at a temperature betweenabout -20° and 0° C. to form the corresponding 5α-chloro thiazolidineamide represented by the above formula C. The reaction is convenientlycarried out in a chlorinated hydrocarbon solvent such as methylenechloride or chloroform and the progress of the reaction can be followedby thin layer chromatography.

The 5α-chloro compound (C) on treatment under anhydrous conditions witha strong base such as sodium hydride or1,5-diazabicyclo[5.4.0]-undec-5-ene (DBU) undergoes cyclization to formthe bicyclic thiazolidine-azetidinone represented by the formula 1.

The cyclization to form (1) is carried out at a temperature betweenabout 0° and 30° C. in an inert solvent. Suitable solvents include thosepreviously mentioned in connection with the foregoing reactions, forexample, the halogenated hydrocarbon solvents such as chloroform,methylene chloride, and trichloroethane. The product (1) of thecyclization is best purified for use in this process by chromatographyover silica gel. Gradient elution employing a gradient of benzene tobenzene-ethyl acetate (7:3, v:v) is a suitable chromatographic systemfor the purification of compound (1).

The 3-acylthiazolidine-4-carboxylic acid is prepared by heating excessacetone or diethyl ketone with L-cysteine at the reflux temperature.

In a preferred embodiment of this invention, thethiazolidine-azetidinone represented by the foregoing formula 1 whereinR is phenyl, each of R' is methyl, and R₁ and R₂ are both benzyl, isreacted with mercuric acetate in tetrahydrofuran containing 50 percentby volume of water to form the oxazoline-azetidinone represented by theformula 2 wherein R is phenyl and both R₁ and R₂ are benzyl. Theoxazoline-azetidinone is chlorinated at about 20°-25° C. with phosphoruspentachloride in methylene chloride in the presence of pyridine to formthe correspondingly substituted3β-(α-chlorobenzylideneamino)-4α-chloroazetidin-2-one represented by theformula 3 wherein R is phenyl and R₁ and R₂ are both benzyl. Thedichloro product is reduced in toluene at about 70° C. withtri(n-butyl)tin hydride and azobisisobutyronitrile to provide thedeschloro reduction product. Without purification the crude deschlororeduction product,1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-benzylideneaminoazetidin-2-one,is dissolved in ethyl acetate and the solution treated withp-toluenesulfonic acid monohydrate to precipitate as thep-toluenesulfonate salt,1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-onerepresented by the formula 5 wherein both R₁ and R₂ are benzyl. Thetosylate salt of the nucleus ester is treated in ethyl acetate with adilute aqueous solution of sodium bicarbonate to form the 3β-aminonucleus ester represented by the following formula ##STR12##

The 3β-aminoazetidin-2-one ester represented by the formula 5 possessesa center of asymmetry at the α-carbon attached to the nitrogen atom ofthe azetidine ring. The compound in the D-configuration is preferred.Accordingly, in carrying out the process of this invention, thethiazolidine-azetidinone starting material represented by the formula 1having the D-configuration is employed.

During the preparation of the thiazolidine-azetidinone starting materialas described above, the cyclization reaction of C to form the bicyclicthiazolidine-azetidinone (1), epimerization can occur. For example, the2,2-dialkyl-3-acyl-5-chlorothiazolidine-4-carboxamide (C) prepared withD-phenylglycine affords C having the D-configuration. Cyclization of Cto 1 is accompanied by epimerization at the asymmetric center resultingin the preparation of (1) as a mixture of D and L isomers. The desiredD-isomer can be separated from the L-isomer by fractionalcrystallization. For example, a solution of the mixture of isomers inethyl acetate on cooling and standing first deposits crystals of theless soluble D-isomer and the filtrate on dilution with petroleum etheraffords a crystalline precipitate of the L-isomer.

The process of this invention provides the hydroxy-protected andesterified nocardicin nucleus 5, which is useful in the synthesis ofnocardicin. Accordingly, the 3β-aminoazetidin-2-one ester 5 is acylatedwith an amino-protected ester of4-(D-3-amino-3-carboxypropoxy)phenylglyoxylic acid O-acyl oxime to formthe amino-, carboxy and hydroxy-protected nocardicin as illustrated inthe following reaction scheme. ##STR13##

R₆ represents a carboxylic acid-protecting group which is readilyremovable under acidic conditions for example diphenylmethyl, benzyl,4-methoxybenzyl, 2,4,6-trimethylbenzyl, or phthalimidomethyl; R₇represents an amino-protecting group for example, the t-butyloxycarbonylgroup; R₁ and R₂ have the same meanings as previously defined herein;and R₈ is acetyl, chloroacetyl or dichloroacetyl.

The above depicted acylation to form the FR 1923 precursor can becarried out by coupling the glyoxylic acid O-acyl oxime with the free3β-amino nucleus compound (5) with a condensing agent such as acarbodiimide or by forming a mixed anhydride of the acid and reactingthe anhydride with the 3β-amino nucleus in the presence oftriethylamine.

The preferred acylation method is the former wherein the acid iscondensed with the amine nucleus with the aid of a condensing agent. Forexample, the 3β-amino nucleus ester (5) is reacted in an inert solventsuch as methylene chloride or tetrahydrofuran with the amino-protectedand carboxy-protected phenylglyoxylic acid O-acetyl oxime in thepresence of an equimolecular amount or a small excess of a carbodiimidesuch as dicyclohexylcarbodiimide. The reaction mixture is maintainedsubstantially anhydrous for best results. The reaction is carried outwith stirring at about room temperature. After the reaction is complete,the insoluble dicyclohexylurea is filtered and the protected nocardicinis recovered from the filtrate.

The protected nocardicin is deblocked to provide nocardicin. Forexample, the protected nocardicin of the above formula wherein R₁ and R₂are benzyl, R₆ is diphenylmethyl, R₇ is the t-butyloxycarbonyl (BOC)amino-protecting group, and R₈ is acetyl, is first reacted withtrifluoroacetic acid at about room temperature to effect the removal ofthe diphenylmethyl ester group R₆, the BOC group R₇, and the O-acetylgroup of the oxime. Thereafter the benzyl groups R₁ and R₂ are removedby treatment of the partially de-blocked molecule with aluminum chloridein an inert solvent containing anisole.

Alternatively the acylation can be carried out with a mixed anhydride ofthe phenylglyoxylic acid. Suitable mixed anhydrides can be prepared withmethyl chloroformate or isobutyl chloroformate. The acylation of theamino nucleus ester (5) is carried out at about 5° to about 25° C. withstirring in a suitable solvent such as methylene chloride ortetrahydrofuran in the presence of a tertiary amine preferablytriethylamine. The reaction is carried out under substantially anhydrousconditions.

The acylation product is next converted to the oxime via reaction withhydroxylamine hydrochloride in an inert aqueous solvent in the presenceof a hydrogen halide acceptor to provide the esterified andamino-protected nocardicin. Following the formation of the oxime, theester groups R₆ and R₁, the amino-protecting group R₇, and thehydroxyl-protecting group R₂ are removed to provide the antibioticnocardicin.

The amino-protected and esterified phenylglyoxylic acid is prepared bythe method described in co-pending application Ser. No. 739,160, filedNov. 5, 1976. As described therein an amino-protected salt ofD-methionine of the formula ##STR14## for example the salt wherein M isdicyclohexylammonium and R₇ is as previously defined herein, isconverted to the trimethylsilyl ester and is alkylated on the sulfuratom with an alkyl or benzyl iodide, for example methyl iodide and thealkylsulfonium iodide of the formula ##STR15## is reacted in an inertsolvent with potassium t-butoxide to form the cyclic amino-protectedD-homoserine lactone of the formula ##STR16## The lactone is hydrolyzedwith an alkali metal hydroxide to the amino-protected D-homoserinealkali metal salt of the formula ##STR17## wherein M' is sodium orpotassium, and the latter is esterified e.g., with diphenylmethylbromide. The esterified D-homoserine is then coupled with a4-hydroxyphenylglyoxylic acid ester, for example, the p-nitrobenzylester, the coupling reaction being carried out with a trialkyl ortriarylphosphine, and preferably triphenylphosphine, and diethylazodicarboxylate to form the amino-protected diester of the formula##STR18## The p-nitrobenzyl ester group is selectively de-esterified byreduction methods whereby the other ester R₆, which is selected fromamong the acid-labile ester groups, remains substantially intact. Forexample, the p-nitrobenzyl ester group is removed via reduction withsodium sulfide. The ester group R₆ which is an acid sensitive group suchas the diphenylmethyl group remains unaffected under the reductionconditions. The selective de-esterification product, the phenylglyoxylicacid, is represented by the formula ##STR19##

In a further aspect of this invention the oxazoline-azetidinoneintermediate (2) is prepared by reacting a3-acylamino-4α-acetoxyazetidin-2-one ester represented by the formula##STR20## in an inert solvent with hydrogen chloride.

In the foregoing formula R, R₁, and R₂ have the same meanings as definedhereinabove.

The reaction is carried out at a temperature between about -10° C. andabout 25° C., preferably at about 0° C. Inert solvents which can be usedare the halogenated hydrocarbon solvents such as chloroform, methylenechloride, dichloroethane, and trichloroethane.

The reaction is carried out by passing hydrogen chloride into a solutionof the 3-acyl-4α-acetoxyazetidinone ester until excess hydrogen chlorideis present. Generally, the solution is saturated with the gas for bestresults. Preferably, the reaction is carried out at about 0° C. to about5° C.

The oxazoline-azetidinone product is recovered by conventional isolationprocedures. For example, the reaction mixture is evaporated to drynessand the residue containing the crude product is dissolved in ethylacetate. The solution is washed with a dilute base such as a dilutesolution of sodium bicarbonate, is dried and optionally treated withcarbon, and evaporated to dryness to obtain the oxazoline-azetidinone.

In a preferred embodiment of this aspect of the invention, the3-benzoylamino-4α-acetoxyazetidinone represented by the above formulawherein R is phenyl, and R₁ and R₂ are both benzyl is dissolved inmethylene chloride and the solution is saturated with hydrogen chlorideat a temperature of 0° C. to provide the oxazoline-azetidinonerepresented by the formula 2 wherein R is phenyl and R₁ and R₂ are bothbenzyl.

The 3-acylamino-4α-acetoxyazetidinone ester represented by the foregoingstructural formula is prepared according to the method described byco-pending application Ser. No. 753,980 filed Dec. 23, 1976. Asdescribed therein a thiazolidine-azetidinone represented by the aboveformula (1) is reacted in acetic acid with mercuric acetate to form a3-(N-propenylacylamino)-4α-acetoxyazetidinone ester represented by theformula ##STR21##

The N-propenylamide is then reacted in a water miscible solvent with adilute mineral acid such as dilute hydrochloric acid to effect thehydrolysis of the propenyl group to provide the3-acylamino-4α-acetoxyazetidinone ester.

In a further aspect of this invention, certain novel compounds areprovided which are useful in the process of this invention for thesynthesis of nocardicin. One group of such compounds are the previouslydescribed oxazoline-azetidinones represented by the formula 2. Preferredcompounds are represented when in the formula 2, R is phenyl, R₁ isbenzyl or diphenylmethyl, and R₂ is benzyl or diphenylmethyl. Anespecially preferred compound is represented by the formula 2 when R isphenyl and both R₁ and R₂ are benzyl.

Another group of novel compounds which are useful intermediates in theprocess of this invention are the dichloro and deschloroazetidin-2-onesrepresented by the formula ##STR22## wherein R, R₁, and R₂ have the samemeanings as previously defined herein and Z and Z' are both hydrogen orchloro.

Examples of compounds represented by the above formula are1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-benzylideneaminoazetidin-2-one,1-[α-(diphenylmethoxycarbonyl)-4-diphenylmethoxybenzyl]-3β-benzylideneaminoazetidin-2-one,1-[α-(methoxycarbonyl)-4-benzyloxybenzyl]-3β-ethylideneaminoazetidin-2-one,1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-phenethylideneaminoazetidin-2-one,1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-(α-chlorobenzylideneamino)-4α-chloroazetidin-2-one,and1-[α-(diphenylmethoxycarbonyl)-4-diphenylmethoxybenzyl]-3β-(.alpha.-chlorobenzylideneamino)-4α-chloroazetidin-2-one.

Preferred compounds of the above formula are those having theD-configuration. A further preferred group is represented when R isphenyl and R₁ and R₂ are benzyl or diphenylmethyl. An especiallypreferred intermediate of the above formula is represented when R isphenyl and both R₁ and R₂ are benzyl.

The following examples are provided to further illustrate the process ofthis invention and the preparation of the intermediates useful therein,and are not intended to limit the scope of this invention.

The abbreviations used in the examples refer to the following: φ=phenyl,TLC=thin layer chromatography, TMS=tetramethylsilane,BOC=-t-butyloxycarbonyl, T60=Varian Associates Model T60 NuclearMagnetic Spectrometer, and in the description of the nuclear magneticspectra, s=singlet, d=doublet, m=multiplet, q=quartet, and t=triplet.

EXAMPLE 1 Preparation of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one

To a stirred solution of 2.29 g. of2-benzoyl-3,3-dimethyl-7-oxo-α-[4-(benzyloxy)phenyl]-4-thia-2,6-diazabicyclo[3.2.0]heptane-6-aceticacid benzyl ester in 230 ml. of tetrahydrofuran and 230 ml. of waterwere added 4.58 g. of mercuric acetate. The mixture was stirred at roomtemperature for three hours and was filtered with the aid of talc. Thefiltrate was extracted with ethyl acetate, the extract dried, treatedwith carbon, and then was evaporated to provide the reaction product asa crude oil. The oil was dissolved in the minimum amount of diethylether and the solution was seeded with a few crystals of the product.The product,7-oxo-3-phenyl-α-[4-(benzyloxy)phenyl]-4-oxa-2,6-diazabicyclo[3.2.0]-hept-2-ene-1-aceticacid, benzyl ester, crystallized from solution. The product was filteredand dried. The yield of first crop product was 1.03 g. while anadditional 0.24 g. of product contaminated with a residue was obtainedfrom the filtrate. The NMR spectrum of the first crop of product was inagreement with the following structural formula of theoxazoline-azetidinone product. ##STR23##

NMR (CDCl₃, TMS): 5.00 (s, CH₂); 5.20 (s, CH₂), 5.30 (d, CH), 5.50 (s,CH), 6.30 (d, CH) and 6.70-7.75 (m, aromatic H) delta.

The oxazoline-azetidinone product (500 mg., 0.95 mmole of first cropmaterial) was dissolved in 100 ml. of dry methylene chloride maintainedunder nitrogen and 600 mg. (2.90 mmole) of phosphorus pentachloride wereadded to the solution with stirring. Next, 0.23 ml. of pyridine wereadded and the reaction mixture was stirred at room temperature for 1.5hours. The reaction was followed by TLC on silica gel usingbenzene:ethyl acetate (7:3, v:v). After 1.5 hours almost all of thestarting material had reacted and the product occurred on the TLC asfaster moving material having an Rf of about 0.9.

The reaction mixture was cooled in an acetone-dry ice bath and pouredinto ice cold brine. The methylene chloride layer was separated, washedwith cold brine, dried, treated with carbon and then evaporated to yield480 mg. of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-(α-chlorobenzylideneamino)-4α-chloroazetidin-2-oneas a white foam. The NMR spectrum of the product was in agreement withthe structural formula of the product. ##STR24##

NMR (CDCl₃, TMS): 5.02 (s, CH₂), 5.24 (s, CH₂), 5.36 (s, CH), 5.42 (d,CH), 5.58 (d, CH), and 6.82-8.08 (m, aromatic H) delta.

The dichloro product (480 mg., 0.845 mmole) was dissolved in 10 ml. offreshly distilled dry toluene and 0.423 ml. (1.69 mmole) oftri-(n-butyl)tin hydride and 280 mg. (1.69 mmole) ofazobisisobutyronitrile were added. The reaction mixture was stirred forabout one hour at a temperature of about 70° C. The mixture was cooledand was diluted with ethyl acetate. The mixture was then washedsuccessively with an aqueous solution of sodium bicarbonate, brine, andwater and was dried. After treatment with carbon, the mixture wasevaporated to dryness and the residue triturated with petroleum etherand filtered. The crude residue (485 mg.) was dissolved in toluene andrefrigerated. A crystalline impurity was filtered and the filtrate wasevaporated to dryness to yield 350 mg. of the des chloroazetidinonerepresented by the following formula. ##STR25##

NMR (CDCl₃, TMS): 3.31 (q, CH), 3.98 (t, CH), 4.98 (s, CH₂), 5.17 (s,CH₂), 4.80 (q, CH), 5.67 (s, CH), 6.80-7.80 (m, aromatic H), and 8.33(s, CH) delta.

The des chloroazetidinone product (350 mg.) was dissolved in 100 ml. ofethyl acetate and the solution as shaken vigorously with 30 ml. of 1Nhydrochloric acid. The organic phase was separated, dried, treated withcarbon and evaporated to dryness. The residue was triturated withpetroleum ether and filtered to yield1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-onehydrochloride.

The hydrochloride salt was dissolved in ethyl acetate and the solutionwas shaken vigorously with an aqueous solution of sodium bicarbonate.The organic layer was separated, dried, treated with carbon, andevaporated to dryness. The residue was dissolved in benzene andchromatographed twice over silica gel with a benzene → ethyl acetategradient to yield 100 mg. of the 3β-aminoazetidinone.

NMR (CDCl₃, TMS): 1.67 (s, NH₂), 2.80 (m, CH), 3.86 (m, CH), 4.21 (m,CH), 5.06 (s, CH₂), 5.18 (s, CH₂), 5.59 (s, CH), and 6.80-7.40 (m,aromatic H) delta.

EXAMPLE 2 Preparation of Nocardicin

To a solution of 100 mg. (0.24 mmole) of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one in 10ml. of dry methylene chloride were added 142 mg. (0.24 mmole) of4-[3-(t-butyloxycarbamido)-3-(diphenylmethoxycarbonyl)propoxy]phenylglyoxylicacid O-acetyloxime and 49.5 mg. (0.24 mmole) of dicyclohexylcarbodiimideand the solution was stirred for 4 hours at room temperature. Thereaction mixture was filtered, evaporated in vacuo and 60 mg. of theproduct were isolated by preparative thin layer chromatography oversilica gel using benzene:ethyl acetate, 1:1, v:v. The acylation productis represented by the following formula. ##STR26##

The acylation product, 60 mg., was dissolved in 2 ml. of trifluoroaceticacid and after the solution was shaken for 2 minutes at roomtemperature, it was evaporated in vacuo. The residue was triturated withdiethyl ether and 36 mg. of the partially deblocked product,3β-[4-(3-amino-3-carboxypropoxy)-2-hydroximino-2-phenylacetamido]-1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]azetidin-2-one,were obtained.

The partially deblocked product, 36 mg., was dissolved in 2 ml. of drymethylene chloride with stirring and the solution was cooled to atemperature of about 0° C. A mixture of 51 mg. of aluminum chloride(0.045 mmole), 48 mg. of anisole (0.415 mmole) and 2 ml. of nitromethanewas added dropwise to the cold solution. After 15 minutes the reactionmixture was allowed to warm to room temperature and the methylenechloride and nitromethane were evaporated off in vacuo at roomtemperature. Water (14 ml.) was added to the concentrate and the pHadjusted to 6.9 with an aqueous solution of sodium bicarbonate. Theaqueous solution was desalted via column chromatography over charcoal(Pittsburgh 12-40 mesh). The column was first eluted with 100 ml. ofwater to collect fractions 1-14 and then with 200 ml. ofwater:acetone:ammonium hydroxide, 100:100:1, v:v, to collect fractions15-50. The fractions were lyophilized.

    ______________________________________                                        Fractions    Weight      Product                                              ______________________________________                                         1-14        135 mg.     salts                                                16-20         8 mg.      nocardicin                                           21-50         9.5 mg.    impure nocardicin                                    ______________________________________                                    

Bioautographs run with the nocardicin product obtained showed theproduct to be identical with authentic nocardicin. The detectingmicroorganisms used on the bioautographs were Serratia marcescens andBacillus steriothermophilus.

EXAMPLE 3 Alternate preparation of oxazoline-azetidinones in aqueousmethanol

A suspension of 500 mg. of the thiazolidine-azetidinone,2-benzoyl-3,3-dimethyl-7-oxo-α-[4-(benzyloxy)-phenyl]-4-thia-2,6-diazabicyclo[3.2.0]heptane-6-aceticacid, benzyl ester, in wet methanol was heated on the steam bath untilsolution was obtained. Mercuric acetate (600 mg.) was added to thesolution and heating was continued for 10 minutes on the steam bath. Theprecipitate which formed was filtered through filter aid, the filtratewas evaporated to dryness in vacuo and the residue dissolved in ethylacetate. The solution was washed with aqueous sodium bisulfite and dueto the formation of an emulsion, the mixture was filtered through filteraid. The organic layer was separated from the aqueous layer and waswashed with aqueous sodium bisulfite and with brine and was then dried,treated with carbon, filtered, and the filtrate evaporated to yield 447mg. of crude oxazoline. The crude product was purified via preparativethin layer chromatography on silica gel plates employing benzene:ethylacetate (7:3, v:v) for development and 263 mg. of purified oxazoline,7-oxo-3-phenyl-α-[4-(benzyloxy)-phenyl]-4-oxa-2,6-diazabicyclo[3.2.0]hept-2-ene-1-aceticacid, benzyl ester, were obtained.

EXAMPLE 4 Alternate preparation of oxazoline-azetidinone via3-acylamino-4α -azetidin-2-one

A solution of 222 mg. of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-benzoylamino-4.alpha.-acetoxyazetidin-2-onein 150 ml. of methylene chloride was cooled to a temperature of about 0°C. and hydrogen chloride was bubbled into the solution until saturationwas achieved. The reaction mixture was evaporated under reduced pressureand the residue dissolved in ethyl acetate. The ethyl acetate solutionwas washed with a dilute aqueous solution of sodium bicarbonate, wasdried, and then evaporated to dryness to yield 200 mg. of7-oxo-3-phenyl-α-[4-benzyloxyphenyl]-4-oxa-2,6-diazabicyclo[3.2.0]hept-2-ene-1-aceticacid, benzyl ester.

EXAMPLE 5 Alternative preparation of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one

To a solution of 480 mg. (0.84 mmole) of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-(α-chlorobenzylidene)amino-4α-chloroazetidin-2-oneprepared as described by Example 1 in 10 ml. of dry toluene (dried bydistillation over calcium hydride) were added 0.42 ml. (1.68 mmole) oftri-(n-butyl)tin hydride and 278 mg. (1.68 mmole) ofazobisisobutyronitrile. The reaction mixture was stirred for 1 hour at atemperature of about 70° C. The course of the reaction was followed byTLC using benzene:ethyl acetate (9:1, v:v). After one hour the mixturewas diluted with ethyl acetate and was washed successively with anaqueous solution of sodium bicarbonate, brine, and with water, was driedand treated with decolorizing carbon before being evaporated to dryness.The crude residue was triturated with petroleum ether and isopropylalcohol.

The crude product mixture was dissolved in diethyl ether and withstirring hydrogen chloride was passed over the solution. The product,1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-onehydrochloride precipitated from solution. The ether was decanted and theproduct was triturated with ether. The free 3β-aminoazetidin-2-one esterwas sprung from the hydrochloride salt with sodium bicarbonate. Theproduct was purified via preparative TLC (silica gel plates,benzene:ethyl acetate, 1:1) to afford 11 mg. of the nucleus amine of theformula ##STR27##

EXAMPLE 6 Alternate preparation of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one viap-toluenesulfonic acid salt

A solution of 480 mg. (0.845 mmole) of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-(α-chlorobenzylideneamino)-4α-chloroazetidin-2-onein 10 ml. of dry toluene was treated with 0.423 ml. (1.69 mmole)tri(n-butyl)tin hydride and 280 mg. (1.69 mmole) ofazobisisobutyronitrile and the mixture heated at 70° C. for 1.5 hours.The1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-benzylideneaminoazetidin-2-onewas recovered in semi-crude form from the reaction mixture. A third ofthe semi-crude product was dissolved in 5 ml. of ethyl acetate and 57mg. of p-toluenesulfonic acid monohydrate were added to the solution.The solution turned dark yellow immediately. After standing at roomtemperature for about 20 minutes, 65 mg. of1-[α-(benzyloxycarbonyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-onep-toluenesulfonate salt crystallized. The salt was filtered, washed withdiethyl ether, and dried.

Another batch of the above p-toluenesulfonate salt was prepared withadditional semi-crude deschloro 3β-benzylideneaminoazetidin-2-one andwas combined with other batches of the same salt. The combined batchesof tosylate salt (130 mg.) were dissolved in 5 ml. of ethyl acetate andthe solution was shaken vigorously in a separatory funnel with 5 ml. ofa saturated aqueous solution of sodium bicarbonate. The organic layerwas separated, dried, and evaporated yielding 92 mg. of1-[α-(benzyloxybenzyl)-4-benzyloxybenzyl]-3β-aminoazetidin-2-one.

We claim:
 1. The process for preparing a 3β-aminoazetidin-2-one of theformula ##STR28## which comprises the steps of (a) reacting thethiazolidine-azetidinone of the formula ##STR29## at a temperaturebetween about 25° C. and about 100° C. with between about 2 and about 4moles of mercuric acetate per mole of said thiazolidine-azetidinone in awater-miscible organic solvent containing between about 10 percent andabout 60 percent water to form the oxazoline-azetidinone of the formula##STR30## (b) reacting said oxazoline-azetidinone in an inert solvent ata temperature between about -10° C. and about 45° C. with at least a 0.1molar excess of a halogenating agent selected from phosphoruspentachloride, phosphorus trichloride, antimony pentachloride, andantimony trichloride in the presence of a tertiary amine to form thedichloro-3β-iminoazetidin-2-one of the formula ##STR31## (c) heatingsaid dichloro-3β-iminoazetidin-2-one in an inert solvent with about 2moles per mole of said dichloroazetidin-2-one of an organo tin hydrideof the formula ##STR32## wherein R₃, R₄, and R₅ independently are C₁ -C₄alkyl, phenyl, or phenyl substituted by methyl or chloro, in thepresence of azo-bisisobutyronitrile to form the deschloro3β-iminoazetidin-2-one of the formula ##STR33## (d) hydrolyzing in thepresence of an acid said 3β-iminoazetidin-2-one;(e) recovering the saltof said 3β-aminoazetidin-2-one formed with said acid; and (f) feedingsaid 3β-aminoazetidin-2-one from said salt. where in the above formulasR is C₁ -C₃ alkyl, phenyl, or benzyl; both of R' are methyl or ethyl; R₁is methyl, benzyl, 4-methoxybenzyl, or diphenylmethyl; and R₂ is benzyl,4-methoxybenzyl, or diphenylmethyl.
 2. The process of claim 1 where instep (a) the thiazolidine-azetidinone is reacted with mercuric acetatein aqueous tetrahydrofuran.
 3. The process of claim 1 where in step (b)the oxazoline-azetidinone is reacted with phosphorus pentachloride. 4.The process of claim 1 where in step (c) the organo tin hydride istri(n-butyl)tin hydride.
 5. The process of claim 1 where in step (d) the3β-iminoazetidin-2-one is hydrolyzed with p-toluenesulfonic acidmonohydrate.
 6. The process of claim 1 where in step (d) the3β-iminoazetidin-2-one is hydrolyzed with aqueous hydrochloric acid. 7.The process of claim 1 wherein R is phenyl, both of R' are methyl, andR₁ and R₂ are both benzyl.
 8. The process of claim 7 wherein thethiazolidine-azetidinone has the D-configuration.